Abstract PR015: Inhibition of androgen receptor signaling in castrate resistant prostate cancer in association with inhibition of glycolysis by targeting hexokinase 2 activity with pyrrolopyrimidine-based small molecules
Authors: Takuma Uo, Cynthia Sprenger, Ian Sweet, Dustin Maly, Ilsa Coleman, Shihua Sun, Kayode Ojo, Mika Munari, Soojin Kim, Kathryn Epilepsia, Gayani Perera, Wanting Han, Peter Nelson, Ryan Choi, Matt Nguyen, Matt Hulverson, Wesley van Voorhis, Stephen Plymate
Journal: Cancer Research (2023)

Abstract The androgen receptor (AR) is the major driver of prostate cancer (PCa) adenocarcinoma. For PCa cells to proliferate, metabolic programs e.g. glycolysis, lipogenesis, oxidative phosphorylation need to be activated to supply the proliferating cells with key anabolic elements. It has recently been shown that glycolysis and enzymes supportive of this pathway including GLUT 1, hexokinase 2 (HK2), and phosphofructokinase (PFK2) are directly upregulated by the AR in castrate resistant prostate cancer (CRPC). HK2 is of particular interest as a target in PCa since, although it is present in fetal tissues, it is suppressed in adult tissues and its function in normal tissues (formation of glucose 6-phosphate) is maintained by hexokinase 1(HK1); however, HK2 expression is markedly increased in AR-driven CRPC in part by an increase in hypoxia-associated HIF1a. Thus HK2 may be an important target in CRPC. In this study we present results of a small molecules, SGI-1553 and SGI-1676 based on a pyrrolopyrimidine-background developed at the University of Washington, that inhibit growth of AR positive cell lines including LNCaP, LNCaP95, VCaP, and LaPC4 but have no effect on PC-3 and DU-145 AR-negative lines. We show that the SGIs specifically inhibit HK2 activity but do not affect HK1. The effects on glycolysis were demonstrated by the suppression of the ECAR using a SeaHorseFX96 instrument. Metabolic flux assay with D-[U-13C] glucose demonstrated reduction in levels of downstream metabolites dihydroxyacetone phosphate (DHAP), phosphoglycerate (PG), phosphoenolpyruvate (PEP), and pyruvate. Indicating a block at either glucose uptake (GLUT1) or HK. We subsequently used two different radiolabeled glucose analogs [3H] 2DG and [3H] 3-O-methyl D-glucose (3-OMG) to demonstrate SGIs inhibit HK. CRISPr KO assays demonstrated that HK2 not HK1 activity on ATP generation as a measure of activity was suppressed by our SGIs. Since the effects of the SGIs were most marked in AR-positive lines and were demonstrated not to interact directly with AR, we examined downstream AR signaling with a probasin-luciferase reporter assay and showed marked suppression of AR R1881 induced activity in LNCaP cells, p &amp;lt;0.001. RNA seq studies also demonstrated suppression of downstream AR-genes. CHiP-seq studies were done to identify effects on AREs. Since SGI-1553 is orally bioavailable in mice with &amp;gt;24 hr half -life and no toxicity at therapeutic doses (20mg/kg three time a week), we treated mice bearing the LuCaP 35 AR-driven PDX model, or PC-3 and DU-145 AR-negative xenografts. There was marked suppression of LuCaP 35 growth (p&amp;lt;0.001) but no effect on PC-3 orDU-145 growth compared to control. Conclusion: SGIs-1553 and 1676 target glycolysis and suppress AR signaling in AR-driven PCa cell lines and PDX models, in part, by inhibition of inhibition of HK2 activity. SGI-1553 is orally bioavailable with an excellent PK profile and no observed toxicity at therapeutic doses in mice, rats and dogs and thus represents the first in-class therapy to target glycolysis in PCa. Citation Format: Takuma Uo, Cynthia Sprenger, Ian Sweet, Dustin Maly, Ilsa Coleman, Shihua Sun, Kayode Ojo, Mika Munari, Soojin Kim, Kathryn Epilepsia, Gayani Perera, Wanting Han, Peter Nelson, Ryan Choi, Matt Nguyen, Matt Hulverson, Wesley van Voorhis, Stephen Plymate. Inhibition of androgen receptor signaling in castrate resistant prostate cancer in association with inhibition of glycolysis by targeting hexokinase 2 activity with pyrrolopyrimidine-based small molecules [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR015.